Introduction
Germ cells have the ability to produce functional gametes and produce individuals with characteristics better than/similar to ancestors. For research purposes this property plays a key role in biotechnology as these germ cells are either unipotent, multipotent or pluripotent. The multipotency or pluripotency of these cells could be manipulated to produce desired cell lines which could be employed in avian or human industry.
Stage X embryonic stem cells (ESCs) of chicken are playing promising role in biotechnology, pharmaceuticals, tissue engineering, cellular therapy and vaccine production. These are pluripotent stem cells which can differentiate into specialized germ and somatic cells (Sun et al., 2020; Zahoor et al., 2024). cESCs express many characteristics similar to mammalian ESCs such as pluripotency markers i.e., stage specific embryonic antigen-1 (SSEA1) and alkaline phosphatase (AKP) (Xiong et al., 2020). In addition, cESCs can produce teratoma in other species after in-vivo injections and can also produce embryoid bodies (EB) during in-vitro manipulations (Yu, 2012; Corsini et al., 2021; Kondoh, 2024). Moreover, cESCs have less cytoplasm to nucleus ratio which makes them favorable for cellular studies (Zhang et al., 2016). Current studies on cESCs include cytotoxicity evaluation of drugs, production of transgenic birds, vaccine development for human and birds and also serve as basic model for different studies (Han and Park, 2018; Ding et al., 2023). However, the maintenance of undifferentiated cESCs for long time in laboratory conditions is very difficult which limits their use (Aubel and Pain, 2013; Wang et al., 2024).
Like cESCs, primordial germ cells of chicken (cPGCs) have self-renewal property, express pluripotency genes (OCT4, NANOG, SSEA1 etc.) and are governed by identical signaling pathways (Meng et al., 2022; Ibrahim et al., 2024; Ichikawa and Horiuchi, 2023; Liu et al., 2024). Additionally, spermatogonial stem cells (SSCs) and PGCs have the property to differentiate back to ESCs which can produce three germ layers viz. ectoderm, mesoderm and endoderm in a controlled environment (Kimura et al., 2015; Matsui et al., 1992; Azizi et al., 2016; Takashima and Shinohara, 2018; Luan et al., 2014). PGCs is the first germ line produced from ESCs and their bi-potency enable them to differentiate either into oocyte or spermatocytes (Tagami et al., 2017). cPGCs act as model to understand the embryonic development as studies have shown similar migration patterns among other species at very early stages (Grimaldi and Raz, 2020). The detailed use of cPGCs will be discussed later within this review along with its application in avian and human industry.
In regenerative medicine spermatogonial stem cells (SSCs) are becoming popular as they pose less ethical concerns as compared to ESCs and PGCs (Chen et al., 2020; Juliá and Medrano, 2020). SSCs originate from A type of spermatogonial cells and play a key role in production of millions of spermatozoa throughout the life of male animals (Kanatsu-Shinohara and Shinohara, 2013; Kubota and Brinster, 2018). Gene edited mice showed higher incidence of teratomas which were later observed to be generated within the seminiferous tubules (Stevens, 1984). Subsequent studies indicated that these teratomas are mainly due to SSCs. These SSCs were later discovered to possess pluripotency properties and in 2004 embryonic stem cells like cells were developed from mouse testicular cells after treatment with reprogramming factors (Kanatsu-Shinohara et al., 2004). Recent studies on human and murine have indicated that SSCs have the capacity to differentiate into three germinal layers and can be reprogrammed to become embryonic stem cells. These cells can then be manipulated to produce somatic cells, thus broadening the potential applications regenerative medicine (Azizi et al., 2016; Chen et al., 2020; Azizi et al., 2019).
More recently, production of induced pluripotent stem (IPS) cells gave some promising results and no serious ethical allegations were raised (Wilson and Wu, 2015; Omole et al., 2022). Human and mouse somatic cells were initially used to reprogram and generate the IPS cells using OSKM (OCT4, SOX2, KLF2 and c-MYC) or OSNL (OCT4, SOX2, NANOG and LIN28) transcription factors (Takahashi and Yamanaka, 2006; Okita et al., 2007; Yu et al., 2007). Induced pluripotent stem (IPS) cells are the alternative stem cells which could be produced using one’s own somatic cells and then can be processed to differentiate into specific cell types. After their first discovery by Takahashi and Yamanaka (Takahashi and Yamanaka, 2006) in mouse, the IPS cells have been produced in many species (Yu et al., 2007; Bessi et al., 2021; Nagy et al., 2011; Kim et al., 2017; Han et al., 2020). The production of IPS cells from domestic and farm animals can be utilized to improve animal welfare, reproduction, drug interactions, disease resistance, meat and milk quality as well as to understand the basic embryonic development. These areas emphasize the production of induced pluripotent stem cells to meet the demands of increasing population.
A notable advancement in the generation of human and rodent IPS cells has been made but production in other species like chicken is still facing many challenges. The major issues reported with chicken IPS cell production are reliance on external factors, limited propagation, less differentiation capacity, low reprogramming ability and limited development.
Origin of chicken germ cells
Waldeyer discovered the germ cells of chicken in the 19th century which led to the discovery of different types of germ cells and their role in the development of chicken (Waldeyer, 1870) (Fig. 1). Chicken embryonic stem cells are pluripotent stem cells which are typically harvested from blastoderm of fertilized eggs (Duda et al., 2025; Lavial, 2010; Petitte et al., 2004). These cells originate from epiblast of blastoderm which is responsible for the formation of ectoderm, mesoderm and endoderm (Lavial, 2010). At the blastula stage embryo undergoes extensive proliferation which results in the formation of a disclike structure containing pluripotent cells. These cells are the precursors of embryonic stem cells with higher self-renewal and maintenance of undifferentiated state under specific conditions like properties (Lee, 2016). After isolation of cESCs researchers developed protocols to culture them in vitro and deciphered the signaling pathways involved in maintaining their self-renewal property. Wnt/β-catenin and FGF/MAPK signaling pathways were initially discovered (He et al., 2018; Guo et al., 2021; Feng et al., 2025) (Liao et al., 2022).
Fig. 1. Historical background of stem cell research in chicken. (Takahashi and Yamanaka, 2006; Waldeyer, 1870; Hensen, 1876; Roux, 1885; Roux, 1888; Swift, 1914; Spemann and Mangold, 1924; Hamburger and HLJJom, 1951; Meyer, 1960; Douarin, 1969; Pain et al., 1996; van de Lavoir et al., 2006; Lu et al., 2014; Nichols et al., 1998; Niwa et al., 2000; Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003; Matsuda et al., 1999; Niwa et al., 1998; Li et al., 2005; Cartwright et al., 2005).
In vertebrates, two models are generally considered for the formation of PGCs including preformation and epigenesis. In the preformation model germplasm is considered as the key regulator of inheritance as it contains RNAs, and proteins and its asymmetrical partition gives rise to PGCs. This phenomenon has also been observed in amphibians (Šimková et al., 2023), zebrafish (Eno, 2016; Yoon et al., 1997), drosophila (Moore et al., 1998) and Caenorhabditis (Seydoux and Strome, 1999). Contrary to preformation model, epigenetic modifications lead to development of PGCs from somatic cells which give evidence regarding the absence of germ plasm in urodele amphibians (Johnson et al., 2003) and mammals (Tam and Zhou, 1996). Origin of avian PGCs have been supported by several studies related to preformation model which is mainly based on the identification and localization of vasa gene in the germ cells. This vasa gene is specifically expressed in germ cells and has been discovered in germ cells of many species i.e., Xenopus laevis and Drosophila melanogaster (Tagami et al., 2017; Ikenishi, 1998). Tsunekawa and colleagues determined the expression patterns of chicken vasa homolog (CVH) in chicken and found its presence in cleavage furrow (Tsunekawa et al., 2000). Recent studies revealed the contribution of vasa in the development of germ cells in both male and female chicken which gives strong evidence about the preformation model of PGCs in chicken (Ichikawa and Horiuchi, 2023; Taylor et al., 2017). The PGCs in birds originate from germ walls and are present in hypoblast and epiblast. Chicken primordial germ cells were first discovered to be present in the blastoderm of HH stage 2 embryo which were later discovered to emerge from epiblast (Eyal-Giladi et al., 1981). Further studies observed that only central region of blastoderm can generate PGCs (Ginsburg and Eyal-Giladi, 1987; Ginsburg and HJGr, 1989). Urven et., (Urven et al., 1988) used a monoclonal antibody (EMA-1) to identify the cPGCs in germinal crescent and during their migration to gonads. These observations and expressions of germ plasm related gene (DAZL) in different developmental stages of chicken also relate to the preformation model. Huang et al., (Huang et al., 2024) observed the apoptosis and abnormal development of germ cells after the knockdown of DAZL gene. Recent transcriptomic study determined the co-expression of DAZL with other genes which are related to zygote formation and developmental gene activation (Rengaraj et al., 2020). Several studies have supported the preformation model of cPGCs but epigenetic regulations of these cPGCs (discussed later in this review) cannot be overlooked as exact origin of these cells is still unknown (Ichikawa and Horiuchi, 2023).
Spermatogonial stem cells (SSCs) are the foundation of male fertility and are responsible for the production of sperm throughout the lifespan of the male individual. PGCs act as the earliest precursors of the SSCs and these PGCs migrate and colonize the genital ridge which give rise to gonads (Song et al., 2022). In male chicken these PGCs localize in the testes and undergo a cascade of events to become the gonocytes which eventually transform into cSSCs (Nakamura and Kagami, 2013). The migration, colonization and transformation of PGCs into SSCs are strictly regulated by intrinsic and extrinsic factors. For example, migration is guided by the stromal cell derived factor 1 (SDF1) along with its receptor CXCR4 (Yahiro et al., 2024). After colonization of PGCs, the Sertoli cells assist their transformation to gonocytes by providing favorable environment. These gonocytes after a period of quiescence differentiate into SSCs which are regulated by different genes and their epigenetic modifications (Garcia and Hofmann, 2013; Manku and Culty, 2015). The presence of Sertoli cells is crucial for maintaining the ability of self-renewal and differentiation of SSCs during the entire life span.
These stem cells of chicken can be manipulated to understand the molecular mechanisms of pluripotency, development of male and female gonads and spermatogenesis not only in chicken but also in other animals. Genetic engineering and other gene editing technologies can be employed to alter these stem cells of chicken and standardize the technique for other higher animals.
Identification of chicken ESCs, PGCs and SSCs
Chicken germ cells are convenient to study because of their large embryonic size, easy accessibility and availability. Identification of germ cells can assist in deciphering the early development, genetic interactions, tissue regeneration and conservation of beneficial traits in the progeny (Laird et al., 2008). Embryonic stem cells of chicken possess unique property of pluripotency and dome-shaped colony formation. These cells have a high nucleus to cytoplasm ratio and prominent nucleoli with distinct colonial boundaries (Choi et al., 2016). Key surface markers and pluripotency markers aid in identifications of cESCs i.e., PouV, Nanog, Sox2, SSEA-1 and ALP (Ren et al., 2024). Initial study conducted by Soodeen-karamath and Gibbins (Soodeen-Karamath and Gibbins, 2001) showed a lack of PouV gene in chicken which was later proved to be wrong by Lavial et al., (Lavial et al., 2007) as homologous gene (Pou5f1) was identified and its role in maintenance of cESCs was established. Moreover, pluripotent genes can be localized on these cells or are identified through their expression at mRNA and protein levels (Choi et al., 2021). Functional identification of cESCs can also be carried out as these cells have the potential to differentiate into neurocytes, cardiomyocytes and fibroblast during in-vitro maturation (Cao et al., 2021; Van Vliet et al., 2012). Moreover, after injection into chicken embryo, these cells can contribute to somatic tissues and germ cells and hence produce chimeras (Zhang et al., 2013).
Chicken primordial germ cells (cPGCs) are large cells with spherical nuclei and have refractive lipid particles in the cytoplasm (Tagami et al., 2017). The diameter of primordial germ cells ranges between 10 and 20 µm and higher glycogen contents make them distinguishable from erythrocyte during their migration towards genital ridge (Tagami et al., 2017; Jung et al., 2017). Periodic acid Schiff (PAS) which can stain glycogen gives promising results in identification of cPGCs. PAS staining can detect cPGCs after 18-20 h of incubation (Niu et al., 2024). The higher contents of glycogen are indicative of more energy requirements during the migration of these cells. But studies on Japanese quail showed contrary results as PAS staining cannot be utilized to detect PGCs due to lower contents of glycogen (Tagami et al., 2017). Similarly, alkaline phosphatase (AP) staining can be utilized to detect cPGCs as these cells exhibit higher levels of alkaline phosphatase (Gong et al., 2024). Additionally, immunohistochemical staining which stains surface antigens i.e., stage specific embryonic antigen-1 (SSEA-1) and embryonic mouse antigen-1 (EMA-1) can also differentiate PGCs from other cells (Ginsburg and Eyal-Giladi, 1987; Karagenc et al., 1996; Fujimoto et al., 1976). Chicken embryonic gonads were used to produce a monoclonal antibody (2C9) which was later employed to detect male and female cPGCs (MAEDA et al., 1994). However, more recent studies showed that these staining techniques are not specific for cPGCs as these surface antigens and glycogen are also expressed in other embryonic stem cells of chicken (Gong et al., 2024). Moreover, PAS staining is effective in the detection of cPGCs after HH 4 stage (Hamburger and HLJJom, 1951), staging system of chicken development) (Pain et al., 1996). Additional studies have determined an increase in vasa and EMA-1 in cPGCs up to 89 % at 3-3.5 days of incubation. At earlier incubation stage (about day 1) only 24 % of PGCs were found to be vasa and EMA-1 positive. SSEA-1, SSEA-2 and SSEA-3 genes were frequently used to differentiate cPGCs from other germ and somatic cells, however, these genes also show some degree of expressions in other cells. So, their expression cannot be specifically related to cPGCs. Moreover, these genes could not be localized in all cPGCs which suggest that they are not expressed in 100 % of PGCs of chicken (Hamburger and HLJDd, 1992; Lee et al., 2016; Mozdziak et al., 2006; Kim and JYJIJoDB, 2018). These studies do not provide enough and specific identification methods that can be used to detect cPGCs which suggest further investigations be carried out (Table 1).
Table 1. Chicken stem cells and their markers for identification.
| Stem Cells | Markers | Reference |
|---|---|---|
| ESCs | ENS, SSEA1, POUV, NANOG, SOX2, C-MYC | (Pain et al., 1996; Lavial et al., 2007; Van de Lavoir et al., 2006; Acloque et al., 2001; Tang et al., 2017) |
| PGCs | CVH, SSEA1, DAZL, C-KIT, BLIMP1, SOX2, NANOG, POUV | (Ichikawa and Horiuchi, 2023; Lavial et al., 2007; Jung et al., 2017; Gong et al., 2024; Karagenc et al., 1996; Whyte et al., 2015; Zhang et al., 2017; Niu et al., 2024) |
| SSCs | GFRα1, PLZF, THY1, C-KIT, DAZL, NANOG, Integrinα6, Stra8 | (Lavial et al., 2007; Sisakhtnezhad et al., 2015; Whyte et al., 2015; Jung et al., 2007; Shi et al., 2014) |
ESCs; Embryonic stem cells, PGCs; Primordial stem cells, EGCs; SSCs; Spermatogonial stem cells.
Like other germ cells, chicken spermatogonia stem cells also possess some unique morphological properties for their identification. These cells are primarily located in the basal compartment of the seminiferous tubules just adjacent to the basement membrane. Unlike other germ cells, these are small cells with round shapes and have relatively large nucleus (Nguyen et al., 2016). Identification of these cells can help in genetic modification, specie conservation and identification of male fertility markers in chicken and other species (Nakamura, 2017; Yu et al., 2010). cSSCs have germ cell specific markers like, chicken vasa homolog (CVH) a conserved germ cell marker in many species, promyelocytic leukemia zinc finger (PLZF) transcription factor essential for their self-renewal, GDNF family receptor alpha 1 (GFRα1) which plays a critical role in their maintenance, deleted in azoospermia-like (DAZL) RNA binding protein and Nanog homeodomain-bearing transcription factor which help in self-renewal and pluripotency (Sisakhtnezhad et al., 2015; Geng et al., 2025). Moreover, cSSCs can be identified and isolated from testicular suspensions using fluorescence activated cell sorting (FACS) based on specific surface markers i.e., GFR α1 and THY1. Similarly, SSC specific promotor (CVH promotor) help in identification of these cells using FACS or fluorescence microscope (Sisakhtnezhad et al., 2015; Sisakhtnezhad et al., 2015). Functional identification of SSCs has been determined through colony formation after supplementation of culture media with GDNF and FGF growth factors and their ability to colonize testes and initiate spermatogenesis after transplantation in the seminiferous tubules of infertile chicken (Li et al., 2008; Rasouli-Gharehsaghal et al., 2020). Identification of these germ cells is essential for studying germ cell development, genetic modification and conservation in case of endangered species.
Isolation of ESCs, PGCs and SSCs
The blastoderm cells of chicken are actually the embryonic stem cells which are similar to mouse embryonic stem cells (mESCs) which give rise to complete individual (Zhang et al., 2013; Eyal-Giladi, 1984). However, cESCs are easily accessible as they are located in the egg which facilitates the isolation of these cells (Lavial, 2010). cESCs can be harvested from stage X embryo after washing of chicken blastoderm with phosphate buffer saline (PBS) and mechanical dispersing (Table 2). Usually, four types of techniques are applied to collect blastoderm cells; 1) paper ring technique is used to separate the embryo with the help of small ring-shaped paper. After placing small paper above the embryonic area of egg yolk, it is cut along the circumference of paper which extracts the embryonic cells attached to it (Horiuchi et al., 2006). This technique is convenient to use, and each embryo extraction requires a separate paper ring. 2) The second method is called spoon method as it requires scooping up of the embryo after cutting from the edges. The careful separation of egg yolk from the embryo is necessary as this method carries out more yolk (Alev et al., 2013; Zhang et al., 2011). 3) The third method is the hair ring method in which the egg yolk is directly cut, and yolk is separated with the help of hair ring (Du and An, 2003; Etches et al., 1997). This method is not suitable at large scale as it requires more precision and is labor intensive. 4) The last known method is the extraction of whole embryos with the help of forceps after cutting them with scissors. Later on, the yolk is stripped off which gives the blastoderm cells or cESCs (Alev et al., 2013). Before collecting the cESCs the eggshells are thoroughly cleaned with alcohol to minimize or completely remove the contaminants. The egg yolk and eggshells are the main sources of contamination and hair pin method is suitable to avoid the contamination as it carries less egg yolk as compared to other methods. Moreover, use of antibiotics i.e. penicillin, gentamicin, kanamycin and streptomycin can prevent contamination (Alev et al., 2013; Du and An, 2003). In contrast to hair ring method, spoon method carries out more egg yolk and hence related with higher chances of contamination. In this case contamination can be minimized with the help of PBS rinsing two to three times. In addition, the embryonic disk of chicken consists of 3 distinct regions viz. zona pellucida, zona opaca and junctional zone. All three zones give different efficiencies in collection of cESCs and the zona pellucida collected cESCs were better in terms of proliferation, growth and maintenance (Xiong et al., 2020; Horiuchi et al., 2006; Kessel and BCJDb, 1986).
Table 2. Collection of Chicken Stem Cells at Different Stages of Development.
| Cell type | Source | Confirmation Methods | References |
|---|---|---|---|
| cESCs | EGK Stage X | Embryoid body formation, somatic chimeras, in-vitro differentiation | (van de Lavoir et al., 2006; Boast and Stern, 2013) |
| cPGCs | HH Stages 14–17 | Chimaeras of germline | (Petitte et al., 2004; Van de Lavoir et al., 2006) |
| cEGCs | HH Stage 28 | Embryoid body formation, somatic chimeras, in-vitro differentiation | (Park and Han, 2000) |
| cGSCs/cSSCs | 6 weeks old or 24 weeks old male roosters | Embryoid body formation, in-vitro differentiation | (Jung et al., 2007; Lee et al., 2006) |
cESCs; Chicken embryonic stem cells, cPGCs; chicken primordial stem cells, cEGCs; chicken embryonic germ cells, cGSCs/cSSCs; chicken gonadal germ cells/chicken spermatogonial stem cells, EGK; Eyal-Giladi and Kochave stage, HH; Hamberger and Hamilton stage.
The isolation and culture of chicken primordial germ cells can be used for genetic modification to produce bird with desired characteristics, for species conservation and for research purposes. Usually, two methods are applied for the isolation of cPGCs viz. isolation from blood circulation at embryonic stage and isolation form gonads. 1) cPGCs from blood circulation are collected between 2.5 to 4.5 days of incubation as after this time these cells start migrating towards gonads. During this procedure a fine glass capillary or micropipette is used to collect the circulating blood from the heart or dorsal aorta of embryo (Fig. 2). After collecting, the blood is mixed with sterile medium either PBS or culture medium. This mixture is later on centrifuged to separate the blood cells from cPGCs (Ibrahim et al., 2024; Lázár et al., 2021). At present there are three centrifugation methods which are commonly used to separate and purify the cPGCs from blood cells. These methods include Ficoll, Percoll and Nycodenz density gradient centrifugation methods. Ficoll density gradient method is the most effective method at removing the blood cells but yields only fewer PGCs. In contrast the Nycodez density gradient method requires preparation of 5 density gradient solutions and gives low purification (Oishi, 2010; Yasuda et al., 1992; Yu et al., 2019). These density gradient methods generally produce 500-2000 PGCs from 20 chicken embryos (Zhang et al., 2023). Specific biomarkers like SSEA-1, DAZL or CVH can be used to identify the cPGCs using FACS or immunostaining (Jung et al., 2017). 2) cPGCs can be collected from gonads after 5.5 to 6 days of incubation as they migrate to the gonads by this time. During gonadal isolation of cPGCs the gonads are collected from the embryo and dissociated with the help of enzymatic digestion (usually, trypsin of collagenase). The cell suspension is filtered to remove the debris and cPGCs are isolated with the help of magnetic activated cell sorting (MACS) or FACS using cPGC-specific antibodies (Nakajima et al., 2023; Tiambo et al., 2021). High purity can be obtained with the help of MACS or FACS as compared to gradient methods, but the yield is very low (about 50 PGCs per embryo). In addition, the cPGCs collected from gonads are at higher maturity levels as compared to blood cPGCs and show differential expressions of genes (Zhang et al., 2023). Another recent technique to isolate the cPGCs from blood is the cell culture insert or CEF adhesion method. In this method cell culture inserts are placed in 24 well plate containing CEFs and collected blood is then added to these culture inserts. CEFs and PGCs are co-cultured using cPGC-culture medium in culture inserts and these culture inserts are removed after migration of PGCs to the lower CEF. Wash the PGCs with PGC culture medium and then transfer to 0.1 % gelatin coated new 24 well plate. Incubate the plate for 12 h and detach the CEFs to purify the PGCs (Zhang et al., 2023).
Fig. 2. Collection of Embryonic Stem Cells, Primordial Germ Cells and Spermatogonial Stem Cells at different stages of incubation. PGCs are collected either directly from circulating blood or from extracted gonads of the chick embryo. ESCs; embryonic stem cells, PGCs; primordial germ cells, HH; Hamberg and Hamilton (Embryonic development stages), SSCs; spermatogonial stem cells.
Similar to cESCs and cPGCs the isolation and culture of cSSCs can be used to study germ cell biology, genetic engineering and avian reproduction. The cSSCs are obtained from testes of chicken embryo or hatched chicken at different days (Ibtisham et al., 2024; Momeni-Moghaddam et al., 2014; Li et al., 2010). Recent study has reported that SSCs with highest viability can be obtained from 21-day old chicken. Moreover, combination of 0.02 g/L EDTA and 0.125 % trypsin gave maximum numbers of viable SSCs (Ibtisham et al., 2024). The method commonly used for the isolation of cSSCs is the extraction of male chicken testes either from embryo or hatched bird. After extraction the testes are washed with sterile PBS and then minced into small pieces. The minced tissue is then digested enzymatically with collagen, trypsin or EDTA to dissociate the cells (Nguyen et al., 2016; Ibtisham et al., 2024). The cell suspension is filtered through a mesh (40-70 um) to remove the debris and then the sediment is then transferred to petri plates containing cSSCs culture medium (Momeni-Moghaddam et al., 2014). FACS or MACS techniques can be utilized to isolate the cSSCs based on their specific biomarkers (THY1, GFRα1 or PLZF) (Sisakhtnezhad et al., 2015; Boozarpour et al., 2016). The collection of cESCs, cPGCs and cSSCs are graphically illustrated in Fig. 2.
Culture of germ cells
The culture of cESCs in laboratory conditions is dependent on several factors. The leukemia inhibitory factor (LIF) plays a critical role in the self-renewal of ESCs as it activates several signaling pathways including phosphoinositide 3 kinase/protein kinase-B (PI3K/Akt), SH2 domain containing tyrosine phosphatase-2/mitogen activated protein kinase (SHP2/MAPK) and Janus kinase/signal transducer and activator of transcription-3 (JAK/STAT3) signaling pathways (Onishi and Zandstra, 2015; Huang et al., 2015; Ye et al., 2016). LIF inhibited (1000 IU/mL of culture medium) the differentiation of cESCs and clones of AKP-positive cESCs were maintained for longer duration in a study conducted by (Du and An, 2003). Similarly, addition of LIF in cESCs culture medium resulted in the activation of STAT3 and expression of ESCs specific markers i.e., SSEA1 and AKP (Zhang et al., 2016; Nakano et al., 2011). Another factor, fibroblast growth factor (FGF) which generates developmental signals and help in cell division in neuroectoderm, and mesoderm was found to maintain the pluripotency of embryonic stem cells. FGF2 receptors mediate the PI3K/Akt signaling pathway along with other signaling pathways to maintain the pluripotency. In humans FGF maintains high levels of Nanog gene and activate PI3K/Akt signaling pathway thus increases survival and proliferation of ESCs (Zhang et al., 2019). In chicken, addition of FGF increased the size and number ESCs colonies as compared to addition of LIF and higher expressions of FGFRs were observed. Moreover, the inhibition of FGFR by PD173074 resulted in partial differentiation of cESCs which shows roles of FGFRs in maintenance of pluripotency (Zhang et al., 2018). Another set of growth factors consists of three family members, Activin, bone morphogenic protein (BMP) and transforming growth factor beta (TGFβ). This family plays diverse functions in proliferation, morphogenesis, cell fate determination, apoptosis and differentiation by regulating BMP/growth, Activin/TGFβ/Nodal and Mullerian inhibiting substance pathways (Abou-Ezzi et al., 2019). Moreover, these TGFβ and activin promote the expression of Nanog gene which block the endodermal differentiation signals and hence maintains the pluripotency in ESCs (Bertero et al., 2015). Similarly, BMP inhibits the expression of differentiation genes and maintains the self-renewal of cESCs along with LIF in a culture medium (Hayashi et al., 2016).
The nutrition for growth of germ cells is maintained in vitro with the supplementation of fetal bovine serum as it is a rich source of nutrients and keeps the environment stable during the culture. Serum also provides some adherent factors which help in adherence of cells on plastic plates during culture (Kong et al., 2018; Sakwe et al., 2010). However, addition of serum leads to differential expression of genes which give variable results in maintenance of pluripotency of ESCs as observed by (Ren et al., 2024). Recently, knockout serum replacement (KSR) was developed to culture ESCs of different mammals which gave promising results (Desai et al., 2015). Farzaneh et al., (Farzaneh et al., 2018) observed the generation of cESCs colonies with the use of KSR along with other supplements.
The addition or feeder layers provide bFGF and LIF which inhibits the differentiation of ESCs and enhance self-renewal. Mostly somatic cells are used to prepare feeder layers such as chicken embryo fibroblasts (CEFs) and mouse embryo fibroblasts (MEFs) (Azizi et al., 2019). In a study conducted on duck ESCs the efficiency of both CEF and MEF in the derivation was high but they failed to maintain the pluripotency after first passage (Guan et al., 2010). Miyahara et al., (Miyahara et al., 2016) observed a promising growth of cESCs in CEF feeder layer but it could not prevent the differentiation. In contrast to DF1 feeder layer with other growth factors (LIF, bFGF and mSCF) maintained typical morphology of cESCs, expression of pluripotency markers and a stable proliferation during long term culture (Zhang et al., 2018). The adhesion, proliferation and migration of ESCs were improved with the addition of Matrigel, gelatin and collagen. However, these extracellular matrices (ECMs) did not support self-renewal of cESCs and potentiated their differentiation into 3 germ layers (Farzaneh et al., 2018). The use of either feeder layers or ECMs led to abnormal expression of genes, morphological changes and abnormal colonies development (Lavial, 2010). Moreover, feeder layers and use of serum in cESCs culture medium raise concerns about safety in clinical applications (Mallon et al., 2006). So, the discovery of the most efficient culture medium and factors are still needed to culture the eESCs in laboratory conditions.
The maintenance of cellular homeostasis, pluripotency and genomic stability are the main functions of culture medium. cPCGs as unipotent stem cells require special culture medium and conditions (Dehdilani et al., 2023). The first report of cPGCs culture for longer duration and production of transgenic chicken dates to 2006 (Van de Lavoir et al., 2006) as before that it was quite difficult to culture cPGCs. Several growth factors, culture systems and supplements have been tried to achieve the above-mentioned goals for cPGCs. Maily three feeder layers, sandoze inbred mouse derived thioguanine resistant and ouabain resistant (STO) fibroblast, Buffalo Rat Liver (BRL) cells and chicken embryo fibroblast (CEF) cells are used to culture cPGCs in vitro (Van de Lavoir et al., 2006; Oishi et al., 2016; Naito et al., 2015; Szczerba et al., 2020). However, contamination of growth media and cPGCs is commonly seen due to these animal derived feeder layers and preparation of these is also laborious and time consuming (Dehdilani et al., 2023). To overcome these problems, feeder-free culture systems are being developed. Whyte et al., (Whyte et al., 2015) observed that FGF2, Activin A and insulin were sufficiently able to culture the cPGCs without the use of any feeder layers. These factors led to the forced expression of Akt, MEK1 and SMAD3 proteins mediated signaling pathways which helped in maintenance of self-renewal and pluripotency of cPGCs. Similar to cESCs, both FGF and TGFβ signaling are required for cPGCs which maintain their proliferation and self-renewal (Van de Lavoir et al., 2006; Whyte et al., 2015; Choi et al., 2010; Macdonald et al., 2010; Vallier et al., 2005). The role of Activin/Nodal/BMP receptors and signaling pathways led to phosphorylation of SMAD proteins which play crucial role in migration and maintenance of cPGCs were determined in a recent study. In addition, it was found that cPGCs required an osmolality under 300 mOsm/kg for growth and proliferation during in vitro culture. Moreover, Lower levels of calcium (0.15 mM) led to indefinite propagation of both male and female derived cPGCs (Whyte et al., 2015). Several studies have reported the beneficial roles of insulin, Activin and FGF on the proliferation and growth of cPGCs during in vitro maintenance. These factors play the same role for development of cPGCs as mentioned for cESCs as both expresses different degrees of pluripotency. Recently, the use of a functional polymer (FP003) in cPGCs culture media enhanced the utilization of nutrients and space occupancy which consequently improved their expansion (Chen et al., 2018). This polymer increased the number of cPGCs by 17 folds in a week of culture. Another study conducted by Taemeh et al., (Yousefi Taemeh et al., 2021) showed that addition of glutamine in the culture media also led to better proliferation and expansion of cPGCs.
Spermatogonia stem cells (SSCs) possess unique property of unipotency as they differentiate into mature spermatozoa. However, their maintenance, growth and culture require special laboratory conditions due to their quality of unipotency. Most of the growth promotors which enhance proliferation, self-renewal and unipotency are the same as being employed for ESCs and PGCs. The first report on derivation of mammalian SSCs from in vitro culture on STO feeder cells dates to 1998 (Nagano et al., 1998). Since then, many studies have reported the isolation, maintenance and culture of SSCs in mouse, rat, human and farm animals (Ibtisham et al., 2024; Wang et al., 2015; von Kopylow et al., 2016; Vlajković et al., 2012; Guan et al., 2009; Oatley et al., 2016). Among non-Mammalian species the chicken industry attracts the focus of researchers due to their higher impacts on human life quality (Fig. 3). Successful isolation and short-term maintenance of SSCs have been attempted in quail and chicken (Pramod et al., 2017; Jung et al., 2007). However, only limited studies are available on isolation, culture and maintenance of SSCs in avian species. Many studies have determined the crucial roles of GDNF, bFGF and LIF in proliferation, maintenance and self-renewal of spermatogonial stem cells (Gritti et al., 1996; Berger and Sturm, 1997; Meng et al., 2000). These additives are required by the growth and development of different species throughout their life (Kubota et al., 2004; Nagano et al., 2003). The cSSCs were isolated and cultured for the first time by Pain et al., (Pain et al., 1996). In recent study Momeni-Moghaddam and colleagues determined the optimum proliferation of cSSCs with the supplementation of GDNF (15ng/mL), bFGF (20 ng/mL) and LIF (15 ng/mL) in an in vitro condition. Moreover, the stem cell pluripotency marker (OCT4) was observed to be highly expressed in cSSCs which were culture in a medium containing GDNF, bFGF and LIF as compared to culture medium without these additives. They also introduced non-enzymatic detachment procedure for cSSCs as enzymatic detachment is considered a damaging factor because of their negative effect on extracellular and cellular matrixes (Yoon et al., 2013). They mechanically smashed the testicular tissues and then passed them through 70 um pore sized mesh. This procedure was rapid compared to previously used two step digestion methods using trypsin and collagenase in chicken, human and sea animals (Momeni-Moghaddam et al., 2014; Panda et al., 2011; Liu et al., 2011). Several other studies have reported the addition of chicken serum, calf fetal serum, gentamicin sulfate, L-glutamine, β-mercaptoethanol, SCF, bFGF, LIF, IGF, IL-11 and non-essential amino acids which maintained the survival, proliferation and self-renewal ability of cSSCs (Li et al., 2010; Jung et al., 2007; Wang et al., 2023; Sisakhtnezhad et al., 2016). The combinations of these growth factors are necessary as many studies on different animals have reported only limited effects of these when applied alone (Zhang et al., 2016; Rasouli-Gharehsaghal et al., 2020; Kanatsu-Shinohara et al., 2006). Moreover, the use of B27 as a replacement of fetal bovine serum increased the proliferation and self-renewal of cSSCs. This B27 contains biotin, α-tocopherol, recombinant insulin, D-galactose, reduced glutathione, linolenic acid and many other amino acids (Sisakhtnezhad et al., 2016).
Fig. 3. Culture of Germ cells at small and large scales and the production of Chimeric chicken which can produce Monoclonal antibodies/proteins and vaccines. ESCs; embryonic stem cells, PGCs; primordial germ cells, SSCs; spermatogonial stem cells, PAS/ALP; periodic acid-Schiff staining/alkaline phosphatase staining.
Epigenetic regulations of germ cells
Epigenetic regulation plays a crucial role in the growth, maintenance, self-renewal and transfer of genetic material to the next generation in germ cells. The conservation of epigenetic modifications has been observed across many species. However, in cESCs higher numbers of trimethylated histones (H3K27me3) were observed in pericentric heterochromatin (PCH) (Kress et al., 2016). The higher expression of DNA methylated related genes (DNMT3B and SMARCA6) expression were observed in undifferentiated cESCs as compared to differentiated cESCs and fibroblasts (Kress et al., 2016). In contrast, He et al., (He et al., 2018) observed some unmethylated and inactivated genes (IFH2, KLF4 and GDNF) in cESCs as compared to cPGCs which were activated in cSSCs. Few genes with low methylation levels were highly expressed in cESCs and other germ cells of chicken which participate in colorectal, lung and endometrial cancer pathways.
Seisenberger and colleagues (Seisenberger et al., 2012) observed a genome wide genetic redistribution between 8.5- and 13.5-day old embryos in mice. These epigenetic modifications specify the fate of PGCs and erase the somatic cell transformation markers (Tang et al., 2016). The integrity of PGCs has been linked with these necessary epigenetic modifications across many species (Strome and DJNrMcb, 2015). In mammals, inactivation of one of the X chromosomes and monoallelic expression of many of the genes have been observed through DNA methylation (Kim and JYJIJoDB, 2018). Both male and female primordial germ cells have different methylated genes which have been seen during implantation of embryos and X-linked chromosomes (Jang et al., 2013). These studies indicate evolutionary preservation of these epigenetic modifications between mammals and birds. Several studies have deciphered the epigenetic regulations of cPGCs i.e., Yu and colleagues observed a large number of DNA methylation related changes between HH stage 21 and 28 (3.5 and 5.5 days after incubation, respectively) of chicken embryo. These corresponding stages are also related to migration and colony formation of cPGCs in the gonads (Yu et al., 2019). Recent study has observed a DNMT3B dependent DNA methylation pattern in gonadal cPGCs along with expression of DNA methyltransferase family genes (DNMT1, DNMT3A and DNMT3B) (Rengaraj et al., 2011). In addition, Jang et al., (Jang et al., 2013) observed specific DNA methylation pattern in cPGCs of gonads as compared to fibroblast of chicken embryo. The understating of these methylation related modifications is still on its initial steps; however, it has opened the door for the elucidation of sexual differentiation and gametogenesis. Like DNA methylation, several studies have reported the histone modification in cPGCs. Unlike mice, in cPGCs, the histone modifications are largely based on H3K9me3 instead of H3K27me3 suggesting avian specific epigenetic regulations (Kress et al., 2016). A unique phenomenon of transient loss of histone modifications have been observed in PGCs of mice which regain their modifications around E12.5 (Hajkova et al., 2008). Another epigenetic modification, activation of bone morphogenetic protein 4 (BMP4) signaling pathway has been observed through H3K4me2 in cPGCs (Zhang et al., 2021). Additionally, Jung and Colleagues (Jung et al., 2018) observed the regulation of Nanog (germ cell development related transcription factor) through acetylation of H3k9.
Roles of miRNA and piRNAs in the regulations of cPGCs have also been revealed to little extent. In chicken germ cells sequencing analysis have revealed the expression of specific piRNAs and repression of these piRNAs and their regulatory pathways resulted in loss in DNA integrity, indicating critical role of these piRNAs (Rengaraj et al., 2014; Xu et al., 2017). In addition, Lee et al., (Lee et al., 2011) observed suppression of homeobox A1 and NR6A1 in cPGCs through the activation of miR-181-3p. Furthermore, suppression of somatic maturation of cPGCs has been observed through miR-181-3p which controls the meiotic divisions. Another study reported the role of miRNAs in maintenance of cPGCs integrity and regulation of DNA methylation. Like miRNA, lncRNA also controls epigenetic modifications via regulation of transcription and translation as they directly interact with RNAs and proteins (Jiang et al., 2021). Zuo and colleagues identified specific lncRNA of cPGCs and regulation of CVH expression through lncRNA-PGC transcript1 (lncRNAPGCAT-1) which controls cPGCs formation (Zuo et al., 2020). Several studies have characterized the role of lncRNA in regulation of integrity of cPGCs (Zhang et al., 2021; Jiang et al., 2021). DND1, a binding protein of RNA that maintains PGCs, inhibits apoptosis of germ cells and controls somatic cell differentiation of PGCs in vertebrates, mice and zebrafish respectively (Yamaji et al., 2017; Youngren et al., 2005; Weidinger et al., 2003; Gross-Thebing et al., 2017). Recent studies have cloned DND1 homolog in chicken along with its specific expression in germ cells, however, the function of this gene remained unknown in chicken (Aramaki et al., 2009; Aramaki et al., 2007).
He et al., (He et al., 2018) observed a higher levels of DNA methylation in cPGCs as compared to cESCs and cSSCs through methyl-CpG binding domain protein sequencing (MBD-seq) (Fig. 4). They further identified 916 differentially expressed genes between cESCs and cPGCs of which 4.48 % genes were located on Z chromosome and 65.85 % of these genes were downregulated in cPGCs. Similarly, 726 differentially expressed genes were located between cPGCs and cSSCs out of which 82.35 % were upregulated in cSSCs. These results confirm the activation of most of the sex-linked genes in the second stage to regulate the sexual differentiation. In addition, lower mRNA expressions of these sex-linked genes were observed in all cell types which indicate role of DNA methylation in switching on or off these genes.
Fig. 4. Epigenetic Regulations of Chicken Embryonic Stem cells, Primordial Germ Cells and Spermatogonial Stem Cells. Oval shape with Me written inside indicates level of DNA-Methylation. PGCs show the highest levels of DNA methylation than SSCs and ESCs. ESCs; Embryonic stem cells, PGCs; Primordial germ cells, SSCs; Spermatogonial stem cells, LncRNA; Long non-coding RNAs, miRNA; Micro RNAs, piRNAs; Piwi-interacting RNAs. (Modified from He et al., (He et al., 2018)).
Induced pluripotent stem cells
The pluripotent stem cells are divided into three main categories: 1) preimplantation pluripotent stem cells as seen in ESCs and IPS cells of mouse which form three-dimensional dome shaped compact colonies (Rebuzzini et al., 2021). These cells are dependent on leukemia inhibitory factor (LIF) for self-renewal and both XX chromosomes activation in female (Huang et al., 2015). 2) Post implantation pluripotent stem cells are seen in ESCs and IPS cells of human and also in epiblast stem cells of mouse. Colonies of these cells have flat and monolayered cells which mainly rely on fibroblast growth factor and Activin A for self-renewal. These cells have inactivated X chromosomes in female and are unable to form colonies from single cell (Schnerch et al., 2010; Boroviak et al., 2014). In contrast to these stem cells preimplantation stem cells are capable to form colonies from single cell which is a key property for genetic screening and gene targeting (Yang et al., 2019). However, both types of stem cells have some restrictions in their differentiation to all cell types. 3) The extended pluripotent stem cells which were developed from porcine have the potential to give rise to extraembryonic cells in addition to embryonic stem cells i.e. trophectoderm (Xu et al., 2019).
The first pluripotent stem cells described were human embryo stem cells, however, the manipulation of these cells raised ethical concerns and displayed neoplasia and genetic instability (Baker et al., 2016; Turinetto et al., 2017). Induced pluripotent stem (IPS) cells are the alternative stem cells which could be produced using one’s own somatic cells and then can be processed to differentiate into specific cell types. After their first discovery by Takahashi and Yamanaka (Takahashi and Yamanaka, 2006) in mouse, the IPS cells have been produced in many species. In mouse IPS cells were produced by embryonic and adult fibroblast cells after introduction of 4 transcription factors (OCT4, SOX2, KFL4 and c-MYC). The success of IPS production was determined by their embryonic stem cell-like properties and specific marker genes. Before discovery of IPS cells nuclear transfer technique was used to reprogram the somatic cells (Wilmut et al., 1998; Cowan et al., 2005). Later on, different factors including OCT3/OCT4 (Nichols et al., 1998; Niwa et al., 2000), SOX2 (Avilion et al., 2003), NANOG (Chambers et al., 2003; Mitsui et al., 2003) were discovered which helped in maintenance of pluripotency in embryonic stem cells. Additionally, STAT3 (Matsuda et al., 1999; Niwa et al., 1998), β-catenin (Sato et al., 2004), KLF4 (Li et al., 2005), E-RAS (Takahashi et al., 2003) and c-MYC (Cartwright et al., 2005) were discovered to maintain long-term stem cells and help in their proliferation in in-vitro.
Many studies are available which have produced IPS cells in chicken utilizing muscle fibroblast (Katayama et al., 2018), feather follicle cells (Kim et al., 2017), embryonic fibroblast (Fuet et al., 2018). These studies have employed OCT4, SOX2, KLF4 and c-MYC (OSKM) reprogramming factors along with NANOG and LIN28 in some of the studies (Fig. 5). IPS cells derived from feather follicle cells could not be passaged beyond 10 passages contrary to other types of cells which were successfully passaged to 20-50 passages (Kim et al., 2017; Fuet et al., 2018; Dai et al., 2014; Rosselló et al., 2013). Most of the reports demonstrated that chicken IPS cells could produce embryoid bodies (EBs) or can directly be reprogrammed to differentiate into neuronal lineage (Dai et al., 2014). Moreover, marker genes of different germ layers in embryoid body were detected and IPS cells of chicken also showed expression of these marker genes or viral genes (Kim et al., 2017; Katayama et al., 2018; Fuet et al., 2018; Lu et al., 2014) except one of the studies conducted by Rossello et al., 2013 which showed silencing of transgenes. The injection of chicken IPS cells in X stage chick embryos resulted in the production of chimeras for many tissues and an increased mortality rate was observed with chimerism due to IPS cells injection (Rosselló et al., 2013). Other studies have found the generation of gonads, brain, liver and heart tissues from injected IPS cells in chicken (Kim et al., 2017; Yu et al., 2014).
Fig. 5. Reprogramming of Chicken Somatic and Gonadal Cells to Produce Induced Pluripotent Stem Cells Along with Embryonic Pluripotent Stem Cells. Embryonic Pluripotent Stem Cells/IPSCs can differentiate into embryonic germ layers and gonads. CEF; chicken embryo fibroblast, PGCs; primordial germ cells, SSCs; spermatogonial stem cells, FFCs; feather follicular cells, OCT4; octamer binding transcription factor 4, KLF4; Krüppel-like factor 4, NANOG; nanog homeobox, c-MYC; Cellular myelocytomatosis gene, SOX2; SRY-Box Transcription Factor 2, LIN28; Lin-28 homolog A.
Feather follicles contain stem cells in the epidermis, and these cells are known as label retaining cells. These cells show stem cells like properties and maintain homeostasis and epidermis formation. Furthermore, feather follicle cells show gene and protein expression just like stromal cells and are easy to manipulate to produce pluripotent stem cells. These cells on induction of pluripotency can be differentiated into several types of cells.
Applications of germ cells
Therapeutic protein and antibody production
The increasing demand for recombinant proteins and antibodies to cure metabolic disorders and cancer has shifted the focus of research industry towards finding the efficient and effective methods for their mass production. Many transgenic animal species can produce and secrete these antibodies in their urine, egg or milk. However, transgenic chicken is very favorable and conducive for this purpose due to easy manipulation, short breeding-time, high production and simple management (Ma and MJItAE, 2021; Houdebine, 2009). Additionally, chicken antibodies and recombinant proteins isolated from chicken egg undergo similar post-translational modifications as required for human use to produce drugs (Kojima et al., 2014). Several studies have reported the use of cPGCs to produce human therapeutic proteins in chicken egg (Lillico et al., 2007). A standard sized chicken egg contains about 4 grams of protein and more than 50 % of this is produced due to the activity of ovalbumin gene (Lechevalier et al., 2007; Win, 2016). Only manipulation of this ovalbumin gene through insertion of exogenous genes can produce large quantities of proteins of interest without any damage (Fig. 3). Human interferons, erythropoietin and avian recombinant proteins have successfully been produced in avian egg white (Kwon et al., 2018). Similarly, an anti-cancer protein against CD20 cancerous protein has been produced in eggs of transgenic chicken which was further validated in cases of lymphoma (Kim, 2018). Separation of these recombinant proteins from egg white of chicken is quite easy as compared to separation from secretions of other animals (Xiong et al., 2020).
Vaccine production
The production of vaccine faces many challenges as the current available methods give low titer, low efficiency and high cost for the mass production of vaccines. Avian cell lines provide alternative to these less efficient methods as these cell lines provide flexible and simple systems for rapid vaccine production (Farzaneh et al., 2017; Giotis et al., 2019). cESCs can maintain virus propagation and provide genomic stability during repeated passaging (Xiong et al., 2020). To date, cESCs derived embryonic 66 (EB66) cells has served for the production of more than twenty types of vaccines such as parainfluenza, influenza, poxvirus and measles. In addition, more than sixty EB66 based vaccines were developed for human and animals (Perugi et al., 2019). A more recent manipulation of chicken somatic cells resulted in the production of induced pluripotent stem cells (IPSCs) which can provide an excellent model for the mass production of vaccines (Farzaneh and MJTUoMSJ, 2020).
Production of live attenuated vaccines requires specific pathogen free embryonated eggs and procurement of these eggs need abundant resources and facilities (Andey et al., 2024). Similarly, during higher demands the manufacturer of other vaccines i.e. avian influenza procures the pathogen free embryonate eggs which limits the production of more than one type of vaccines at the same time. Vero cell lines and MDCK have been extensively used for the production of influenza vaccine, but Newcastle disease vaccine production is largely dependent on pathogen free eggs (Liu et al., 2009; Hussain et al., 2010; Ehrlich et al., 2012). Some other cell lines which were tested to produce Newcastle disease virus include, chicken embryo liver, fibroblast and kidney cell lines (Arifin et al., 2011; Shittu et al., 2016). However, these cells need anchorage and are not easy to manipulate to mass produce the required vaccine (Moran, 1999). Recently, BA3 chicken induced pluripotent stem cell lines were used to propagate the Newcastle disease virus and production of LaSota vaccine. This Newcastle disease is the most sporadic disease in developing countries which leads to significant economic losses. The mortality rate in this disease varies from 0 to 100 %. The BA3 pluripotent stem cells have shorter doubling time (21 h) and can grow in a variety of cell media. After 24 h of induced infection a higher viral titer was observed and these lines were resistant to fowl adenovirus, avian leukosis virus, reticulo-endotheliosis, Marek’s disease virus and some mycoplasma infection. These induced pluripotent cell lines (BA3) showed excellent candidature for Newcastle disease virus propagation and vaccine production due to desirable growing characteristics i.e. in serum free medium and shorter doubling time (Shittu et al., 2016).
Transgenic chicken production
Transgenic birds can provide insight into cell tracking, embryogenesis, species hybridization, early development, and conservation of species (Yu et al., 2014; Kagami, 2016). During production of transgenic chicken, the viral vector carrying the exogenous DNA is directly added to the recipient embryo of X stage. Then these were transferred to blastoderm cells. Due to DNA fragment sizes the germ line transmission did not give promising results till date for the production of transgenic chicken (Collarini et al., 2019). Another non-viral method is to insert a large size foreign DNA directly into the zygote blastoderm. However, this method has its own disadvantages as access and exact location of blastoderm is difficult to determine (Bednarczyk et al., 2018).
A more recent technique is the injection of target genes in cESCs and cPGCs in vitro and then injection of these transgenic cells in the dorsal aorta or sub-germinal cavity of embryo at HH stage 13-14 (Van de Lavoir et al., 2006; Chen et al., 2023; Dehdilani et al., 2022). The migration of these cPGCs containing genes of interest into gonads may give rise to recombinant germ cells (Collarini et al., 2019). The successful generation of chimeras has resulted after the injection of donor cESCs into the recipients (Zhang et al., 2013) (Fig. 3). In addition to above applications, germ cells of chicken (cESCs) can be manipulated to produce required stages of embryo for research purposes. These can include but are not limited to study cellular differentiation, genetic expression, genetic regulation, cloning of genetically superior animals. Moreover, due to higher resemblance in ESCs of chicken and human, cESCs can broaden the application and understanding of human ESCs.
Gene editing and functional genomics
Many techniques are available to insert the gene of interest in a specific set of DNAs. The recent techniques include clustered regularly interspaced palindromic repeats (CRISPR/Cas9) and transcription activator like effector nuclease (TALEN) that can create knockout or knock-in germ cells to produce an individual with special characters (Taylor et al., 2017; Oishi et al., 2016; Park et al., 2014; Park et al., 2014). Oishi and colleagues (Oishi et al., 2016) successfully applied the CRISPR/Cas9 system to mutate the two egg white genes in cPGCs. Ovomucoid and ovalbumin genes manipulation produced transgenic chicken with more than 90 % efficiency. These genes are basically related to the stimulation of allergic reactions in humans.
Conservation of genetic resources
To date millions of known and unknown species have become instinct and no technology is available to regenerate those species even if their fossils are utilized. However, the existing species of mammals and birds could be saved from extinction which have left with only fewer animals on this planet. Germ cells isolation and culture have been attempted not only in mammals and chicken but also in quail, duck, pheasant, turkey and guinea fowl for the purpose of restoring avian species (Liu et al., 2011; Ono et al., 1996; Reynaud, 1969; Wernery et al., 2010; Kang et al., 2008; Van de Lavoir et al., 2012). The germ cells like ESCs, PGCs and SSCs could be cryopreserved and used in future to regenerate any specie that could vanish from the face of the earth. Rare chicken breeds are being manipulated to conserve their genetic diversity and germ cells in case of extinction (Naim and Mishra, 2021).
The functional germ cells development from somatic cells using induced pluripotent stem cell reprogramming can prevent extinction of endangered species (Hayashi et al., 2017; Yoshino et al., 2021). Ben-Nun and colleagues reported the IPSCs generation for the first time from an endangered primate (Mandrillus leucophaeus) and white rhinoceros (Ceratotherium simum cottoni) (Friedrich Ben-Nun et al., 2011). Similarly, induction of pluripotency has also been tried in other endangered animals such as Javan banteng, snow leopard and orangutan (Verma et al., 2012; Ben-Nun et al., 2015; Ramaswamy et al., 2015). Similar to human hair follicular cells, feather follicular cells of chicken have the capability of production of IPSCs under specific conditions which could efficiently restore or conserve the avian species without wasting the embryos (Kim, 2018; Nogueira et al., 2024). The potential applications of chicken stem cell research and induced pluripotent stem cells are elaborated in Fig. 6.
Fig. 6. Potential Applications of Chicken Stem Cells and IPSCs. CRISPR/Cas9; clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9.
Current challenges and limitations of chicken stem cell technology
Based on the available data the stem cell technology of chicken gives a promising future in understanding regenerative medicine, vaccine development and developmental aspects of birds, human, and other mammals (discussed in the next section). However, cESCs, cPGCs, cSSCs and chicken IPSCs are difficult to culture and maintain in vitro as compared to mouse stem cells. For example, the pluripotent stem cells of chicken are considered primed, meaning they are in advanced developmental stages and poorly contribute to sperm or egg formation (Chen et al., 2025; Intarapat and Stern, 2013). Moreover, gene editing, chimera and transgenic birds creation is difficult using these pluripotent stem cells. It is difficult to maintain chicken stem cells in a pure and undifferentiated state and tend to spontaneously differentiate into heterogenous cell types, making it unsuitable for precise genetic manipulations (He et al., 2025). The ultimate goal of chicken stem cell technology is to create transgenic animals that can pass the beneficial traits to their offsprings, however, integration of genes in developing germline is difficult due to primed state of these pluripotent stem cells (Yu et al., 2019). Similarly, after successful production of chimera, it takes approximately 6 months for a chicken to produce offsprings which makes screening for germline transmission slower, expensive, and laborious (Kamihira et al., 2009).
Additionally, the optimal culture and maintenance media for culture of cESCs, cPGCs and cSSCs are not fully established. We mostly rely on undefined and complex factors like specific serum batches, feeder layers and environmental conditions, leading to variability and reproducibility problems between labs (Farzaneh et al., 2017). cPGCs are often used to create transgenic chickens due to their fate to differentiate into functional gametes (egg and sperm). During culture and expansion of cPGCs they lose their migratory ability as they have to compete with recipient PGCs to colonize the gonads and form gametes (Chen et al., 2023). Moreover, the annotation of chicken genome is not complete and refined yet as the mouse and human which makes identifying precise genetic control elements challenging for precise genetic engineering (Wu et al., 2024). Similarly, chicken research field has scarcity of standardized and commercially available reagents (e.g., validated genes, growth factors, specific expression kits, and antibodies) compared to mouse and other lab animals (Yousefi Taemeh et al., 2025). Additionally, chicken as a model species receives less intentions and fundings compared to mammalian models (for human regenerative medicine), which slows the pace of innovation (Nguyen et al., 2024).
Future aspects and conclusion
The future research on chicken ESCs, PGCs, SSCs and IPSCs holds immense potential for advancing biotechnology, agriculture and biomedical sciences. The demands for poultry products continue to rise, driven by increased demands for protein, so, the understanding of insightful manipulation of chicken germ cells will be critical to improve poultry products including eggs and meat. Germ cells, especially PGCs and SSCs which transfer genetic information need deeper research and innovation to optimize breeding, produce resistant breeds, and improve meat and egg quality. CRISPR/Cas9 is one of the most promising and recent technology which can introduce desirable genes and traits to germ cells to produce future generations of chicken with enhanced characters. Moreover, stem cells should differentiate into specific tissue and also have self-destruction mechanisms where required to stop the deleterious effects of their multiplications, i.e., tumor production. Additionally, single cell multi-omics studies of chicken stem cells could enhance our understanding of cell lineage during its differentiation into tissue or organ and reveal the precise molecular steps to ease the genetic manipulation of the cells in other species. Furthermore, preservation of germ cells, i.e. ESCs, PGCs and SSCs could pave the way for genetic diversity and conservation of endangered avian species.
In biomedical sciences chicken germ cells have gained special recognition due to their short reproductive cycle and ease in accessing the embryonic germ cells. Deciphering of chicken germ cells could enhance the understanding of human reproduction and fertility. For example, molecular mechanisms of germ cell differentiation in chicken can act as a model for human germ cell differentiation and development. Moreover, in bioreactor system the use of chickens is increasing to produce therapeutic proteins and vaccines. The manipulation of germ cells results in the production of human recombinant proteins in the eggs of chicken which offers cost-effective and scalable alternative to traditional methods. Future research could expand the applications of germ cell utilization to a wider range of medical treatments. For example, understanding the development of complex organism from a single cell will definitely allow the scientists to manipulate the stem cells at a level where they can sense their environment and respond accordingly i.e., release of insulin should be only after its depletion and high levels of glucose in the blood.
Another frontier is the research on germ cell transplantation and the generation of interspecies chimeras. Recent chimeric studies resulted in the production of chicken and quail chimeras. Similarly, chicken stem cell technology may revolutionize the in vitro organ development by programmed self-assembly of stem cells, bio-printing to produce sophisticated and vascularized functional tissues and production of inter-species chimeras to produce human organs in animals i.e., to produce human-compatible pancreas secreting insulin. Better understanding of cross specie chimeras could be a breakthrough in conservation biology and chicken can act as a surrogate host to conserve the endangered species. Additionally, germ cell studies of chicken can provide insight into evolution of avian species and reproductive strategies. Future studies on single cell sequencing and omics can enhance our understanding of signaling pathways and regulatory networks that govern development of these germ cells.
In conclusion, the exploration of chicken germ cells can revolutionize the poultry industry, agriculture, biomedical sciences and biotechnology. Studies of germ cells have the potential to eliminate human and animal genetic and acquired diseases and disorders. Mastering gene editing technology in chicken can help in editing of desirable genes in other species. With technological advancement, the insight gained from the exploration of chicken germ cells will undoubtedly result in a sustainable, healthy and prosperous human future. Moreover, acceptance of germ cell manipulation and genetic modification by the general population will minimize the hinderance in research as these studies sometimes results in unintended outcomes.
Source: Science Direct
